Specific proteolytic processing in secretory compartments and at the cell surface is required for activation of numerous transmembrane and secreted proteins in eukaryotes. The yeast Kex2 protease is the founding member and prototype of the family of subtilisin-related proprotein processing enzymes--conserved in eukaryotes from fungi to vertebrates - that is responsible for most such processing reactions. These enzymes process prohormones and neuropeptides at clusters of basic residues in the regulated secretory pathway and a variety of proproteins in the constitutive secretory pathway. The first known human Kex2 homologue, furin, is implicated in maturation of growth factors and growth factor receptors, coagulation factors, viral glycoproteins and bacterial toxins. Work in the previous funding periods has given the principal investigator a comprehensive, quantitative and mechanistic view of Kex2 and furin specificity. The role of furin in viral life cycles and maturation of bacterial toxins makes it a possible target for development of broad spectrum anti-viral and anti- microbial drugs. With this in mind, and with the knowledge of substrate specificity of furin and Kex2, the principal investigator has developed a novel family of high-affinity inhibitors of proprotein processing enzymes such as furin and Kex2 based on the polypeptide protease inhibitor eglin c. The principal investigator has identified a novel group of glycosylphosphatidylinositol-anchored cell surface aspartyl processing proteases whose function overlaps that of Kex2. Here the principal investigator undertakes biochemical and genetic analysis of the coordinate physiological roles of these GPI-anchored aspartyl proteases, or "yapsins" along with Kex2. Finally, the advancement of this field has been hampered by the absence of structural information. In collaboration with Dr. Dagmar Ringe at Brandeis University, the principal investigator has previously obtained crystals that diffract to 3A but have been stymied by the inability to obtain derivatives. He will now mount a determined effort to obtain crystals suitable for solving the atomic structures of Kex2 and furin. The specific aims of this proposal are as follows: 1. To develop high-affinity, selective inhibitors of proprotein processing proteases and their use in vivo inhibition, purification and crystallization of processing enzymes. 2. To elucidate the biochemistry and physiology of processing of yeast cell wall proteins by Kex2p and GPI-anchored aspartyl proteases. 3. To achieve Kex2p and furin crystallization to advance determination of their structures.